We thank C.C.R. laboratory members for comments and S. Moskowitz for figure preparation. This work was supported by National Institutes of Health Grants F32GM101761 (A.J.H.) and GM54397 (C.C.R.).

NOTE: Follow all institutional regulations regarding the safe use and disposal of radioactive material, including but not limited to, usage of personal protective equipment, such as gloves, safety glasses, lab coat, and appropriate acrylic shields.
NOTE: The standard buffer consists of 40 mM HEPES-KOH, pH 7.5, 50 mM potassium glutamate, 5 mM DTT, 0.1 mM EDTA (this buffer is pre-made at a 5x concentration) and 0.3 mM of each dNTP. T7 replication proteins are purified as described<sup class="xre...

<ol>
	<li>Kornberg, A., Baker, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DNA replication. , (1992).</li><li>Hamdan, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motors,+switches,+and+contacts+in+the+replisome.">Motors, switches, and contacts in the replisome.</a> Annu Rev Biochem. 78, 205-243 (2009).</li><li>Mendelman, L. V., Notarnicola, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+bacteriophage+T7+gene+4+proteins+in+providing+primase+and+helicase+functions+in+vivo.">Roles of bacteriophage T7 gene 4 proteins in providing primase and helicase functions in vivo.</a> Proc Natl Acad Sci U S A. 89 (22), 10638-10642 (1992).</li><li>Qimron, U., Lee, S. J., Hamdan, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+initiation+and+extension+by+T7+DNA+primase.">Primer initiation and extension by T7 DNA primase.</a> EMBO J. 25 (10), 2199-2208 (2006).</li><li>Kato, M., Ito, T., Wagner, G., Richardson, C. C., Ellenberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+architecture+of+the+bacteriophage+T7+primase+couples+RNA+primer+synthesis+to+DNA+synthesis.">Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis.</a> Mol Cell. 11 (5), 1349-1360 (2003).</li><li>Kusakabe, T., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+4+DNA+primase+of+bacteriophage+T7+mediates+the+annealing+and+extension+of+ribo-oligonucleotides+at+primase+recognition+sites.">Gene 4 DNA primase of bacteriophage T7 mediates the annealing and extension of ribo-oligonucleotides at primase recognition sites.</a> J Biol Chem. 272 (19), 12446-12453 (1997).</li><li>Lee, S. J., Zhu, B., Hamdan, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+sequence-specific+template+binding+by+the+DNA+primase+of+bacteriophage+T7.">Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7.</a> Nucleic Acids Res. 38 (13), 4372-4383 (2010).</li><li>Hernandez, A. J., Lee, S. J., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+release+is+the+rate-limiting+event+in+lagging-strand+synthesis+mediated+by+the+T7+replisome.">Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome.</a> Proc Natl Acad Sci U S A. 113 (21), 5916-5921 (2016).</li><li>Tabor, S., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+residue+in+DNA+polymerases+of+the+Escherichia+coli+DNA+polymerase+I+family+is+critical+for+distinguishing+between+deoxy-+and+dideoxyribonucleotides.">A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.</a> Proc Natl Acad Sci U S A. 92 (14), 6339-6343 (1995).</li><li>Kim, Y. T., Tabor, S., Bortner, C., Griffith, J. D., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+the+bacteriophage+T7+gene+2.5+protein.+A+single-stranded+DNA-binding+protein.">Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein.</a> J Biol Chem. 267 (21), 15022-15031 (1992).</li><li>Frick, D. N., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+primases.">DNA primases.</a> Annu Rev Biochem. 70, 39-80 (2001).</li><li>Lee, S. J., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+lysine+residues+in+the+RNA+polymerase+domain+of+the+gene+4+primase-helicase+of+bacteriophage+T7.">Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.</a> J Biol Chem. 276 (52), 49419-49426 (2001).</li><li>Cao, W., De La Cruz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+full+time+course+analysis+of+nonlinear+enzyme+cycling+kinetics.">Quantitative full time course analysis of nonlinear enzyme cycling kinetics.</a> Sci Rep. 3, 2658 (2013).</li><li>Wang, M. H., Zhao, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+determining+kinetic+constants+of+complexing+inactivation+at+identical+enzyme+and+inhibitor+concentrations.">A simple method for determining kinetic constants of complexing inactivation at identical enzyme and inhibitor concentrations.</a> FEBS Lett. 412 (3), 425-428 (1997).</li><li>Johnson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitting+enzyme+kinetic+data+with+KinTek+Global+Kinetic+Explorer.">Fitting enzyme kinetic data with KinTek Global Kinetic Explorer.</a> Methods Enzymol. 467, 601-626 (2009).</li><li>Johnson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+century+of+enzyme+kinetic+analysis,+1913.">A century of enzyme kinetic analysis, 1913.</a> FEBS Lett. 587 (17), 2753-2766 (2013).</li><li>Biswas, T., Resto-Roldan, E., Sawyer, S. K., Artsimovitch, I., Tsodikov, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+non-radioactive+primase-pyrophosphatase+activity+assay+and+its+application+to+the+discovery+of+inhibitors+of+Mycobacterium+tuberculosis+primase+DnaG.">A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG.</a> Nucleic Acids Res. 41 (4), e56 (2013).</li><li>Sanyal, G., Doig, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+DNA+replication+enzymes+as+targets+for+antibacterial+drug+discovery.">Bacterial DNA replication enzymes as targets for antibacterial drug discovery.</a> Expert Opin Drug Discov. 7 (4), 327-339 (2012).</li></ol>

The authors have nothing to disclose.

The most important factor in performing these experiments is the availability of highly active purified enzyme. During our work with gp4, for example, we found that storage of the purified enzyme in buffer (20 mM potassium phosphate, pH 7.4, 0.1 mM EDTA, 1 mM DTT) containing 50% glycerol at -20 &#176;C leads to a reduction in the specific activity of the preparation over a few months. Therefore we now flash-freeze small aliquots of purified gp4 in 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM TCEP, and 10% glycer...

The replication of DNA is a conserved feature of all cellular life. While the identity and number of replication components vary widely, the general mechanisms are shared across evolution1. Here, we describe gel-based, discontinuous kinetic experiments, using radioactive substrates, aimed at understanding the kinetics of lagging-strand DNA synthesis in vitro by components of the T7 replisome. The replication machinery of bacteriophage T7 is very simple, consisting of only four proteins (the DNA polymerase, gp5, and its processivity factor, E. coli thioredoxin, the bifunctional primase-helicase gp4, and the single-stranded DNA binding protein, gp2.5)2. This feature makes it an attractive model system to study the conserved biochemical mechanisms involved in DNA replication.
Special emphasis is placed on the formation of ribonucleotide primers by T7 DNA primase, a critical step in the initiation of DNA synthesis. In addition, one can also examine the usage of these primers by T7 DNA polymerase or other replication proteins by including them in the reaction mixture. The T7 primase-helicase, gp4, catalyzes the formation of primers for DNA synthesis at specific DNA sequences called primase recognition sites, or PRS, (5&#39;-GTC-3&#39;). The cytosine in the PRS is cryptic, it is essential for recognition of the site, but it is not copied into the product3. The first step in primer synthesis by gp4 involves the formation of the dinucleotide pppAC, which is then extended to a trimer, and eventually to functional tetranucleotide primers pppACCC, pppACCA, or pppACAC depending on the sequence in the template4. These primers can then be used by T7 DNA polymerase to initiate DNA synthesis, which is also a gp4-assisted process5,6. In this regard, the primase domain stabilizes the extremely short tetraribonucleotides with the template, preventing their dissociation, engages DNA polymerase in a manner conducive to securing the primer/template into the polymerase active site7. These steps (RNA primer synthesis, primer handoff to polymerase, and extension) are repeated in multiple cycles to replicate the lagging strand and must be coordinated with the replication of the leading strand.
The assays described here are highly sensitive and can be performed in a moderate time frame. However, they are relatively low-throughput and great care must be exercised in the usage and disposal of radioactive materials. Depending on the speed at which the reaction proceeds, one might employ a rapid-quench instrument to attain samples at time scales amenable for meaningful analysis of reaction time courses in either the steady or the pre-steady state. Recently, we used assays described here to provide evidence of the importance of primer release from the primase domain of gp4 in the initiation of Okazaki fragments. Additionally, we found evidence of a regulatory role for the T7 single-stranded DNA binding protein, gp2.5, in promoting efficient primer formation and utilization8.

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			Tris pre-set crystals 
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			<td height="42" style="height:42px;width:216px;">L-Glutamic acid potassium salt monohydrate</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">G1501&#160;</td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Magnesium chloride hexahydrate</td>
			<td style="width:83px;">Mallinckrodt Chemicals</td>
			<td style="width:119px;">5958-04</td>
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			<td height="21" style="height:21px;width:216px;">1,4-Dithiothreitol</td>
			<td style="width:83px;">J.T. Baker</td>
			<td style="width:119px;">F780-02</td>
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			<td height="63" style="height:63px;width:216px;">Sodium Dodecyl Sulfate</td>
			<td style="width:83px;">MP Biomedicals</td>
			<td align="right" style="width:119px;">811030</td>
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			<td height="63" style="height:63px;width:216px;">(Ethylenedinitrilo) Tetraacetic acid, disodium salt, dihydrate</td>
			<td style="width:83px;">Mallinckrodt Chemicals</td>
			<td style="width:119px;">4931-04</td>
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			<td height="42" style="height:42px;width:216px;">[&#945;-32P] CTP</td>
			<td style="width:83px;">PerkinElmer</td>
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			<td>radioactive, take protective measures</td>
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			<td style="width:83px;">PerkinElmer</td>
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			<td>radioactive, take protective measures</td>
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			<td height="42" style="height:42px;width:216px;">100 mM dATP, dCTP, dGTP, dTTP&#160;</td>
			<td style="width:83px;">Affymetrix</td>
			<td align="right" style="width:119px;">77100</td>
			<td>four dNTPs part of set</td>
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			<td height="21" style="height:21px;width:216px;">100 mM ATP and CTP</td>
			<td style="width:83px;">Epicentre</td>
			<td style="width:119px;">RN02825</td>
			<td>part of NTP set</td>
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			<td height="84" style="height:84px;width:216px;">ssDNA constructs</td>
			<td style="width:83px;">Integrated DNA Technologies</td>
			<td style="width:119px;">N/A</td>
			<td>custom sequences&#160;</td>
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			<td height="63" style="height:63px;width:216px;">methanol&#160;</td>
			<td style="width:83px;">Macron Fine Chemicals</td>
			<td style="width:119px;">3017-08</td>
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			<td height="42" style="height:42px;width:216px;">formamide</td>
			<td style="width:83px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">17899</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">bromophenol blue</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">B8026&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">cylene xyanol&#160;</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">X4126&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">acrylamide</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">A9099</td>
			<td>Toxic</td>
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			<td height="21" style="height:21px;width:216px;">N,N&#8242;-Methylenebisacrylamide</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">M7279&#160;</td>
			<td>Toxic</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Urea</td>
			<td style="width:83px;">Boston Bioproducts</td>
			<td style="width:119px;">P-940</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Boric acid</td>
			<td style="width:83px;">Mallinckrodt Chemicals</td>
			<td align="right" style="width:119px;">2549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rapid Quench-Flow Instrument&#160;</td>
			<td style="width:83px;">Kintek Corp.</td>
			<td style="width:119px;">RQF-3&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kintek Explorer</td>
			<td style="width:83px;">Kintek Corp.</td>
			<td style="width:119px;">N/A</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kaleidagraph&#160;</td>
			<td style="width:83px;">Synergy Software</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
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kinetics of lagging-strand dna synthesis in vitro by the bacteriophage t7 replication proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

The results shown in Figure 1A were obtained as described in step 1 of the protocol, i.e., by manually sampling a primer synthesis reaction catalyzed by gp4 under multiple-turnover conditions. Here, a range of products after gel electrophoresis can be observed by using CTP labeled with 32P at the &#945;-position in primer synthesis reactions (Figure 1A). The labeled precursor, CTP, shows the highest mobility and migrat...

Watch this Scientific Journal Video about Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins at JoVE.com

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

The overall goal of this procedure is to examine the kinetics of primer and lagging strand DNA synthesis. This method can help answer key questions in the DNA replication field, such as how primers form by DNA primase, are transferred to, and utilized by, DNA polymerase. The main advantage of this technique is that it allows the identification of key steps during Okazaki fragment synthesis.Since radioactive materials are used in this experiment, all institutional regulations regarding the safe use and disposal of radioactive materials must be followed. Start by aliquoting two microliters of formamide dye buffer into eight 1.5 milliliter polypropylene tubes, equal to the number of time points to be analyzed. Next, prepare a 20 microliter mixture consisting of buffer, ATP, CTP, CTP labeled on the alpha-phosphate group by P32, single-strand DNA template, and the gene four protein, or GP four primase helicase.Incubate the mixture at room temperature for five minutes. After five minutes, use a pipettor to remove two microliters of the mixture, and mix it with two microliters of formamide dye buffer in one of the 1.5 milliliters tubes prepared earlier. This is the no reaction control.Add two microliters of 0.1 molar magnesium chloride to the reaction mixture and immediately start a timer. At 10 second intervals, manually withdraw two microliter samples of the reaction mixture, and mix with the formamide dye in the 1.5 milliliter tubes. After all time points have been collected, heat the samples in a heat block at 95 degrees Celsius for five minutes.Load aliquots of the samples onto a denaturing gel for electrophoresis. Begin this procedure by preparing the reaction mixtures. Prepare 400 microliters of solution A containing buffer, GP four hexamer, single-strand DNA template, dNTPs, ATP, CTP, and CTP labeled on the alpha-phosphate group by P32.Prepare 400 microliters of solution B, containing one X buffer and 20 millimolar of magnesium chloride. Next, set up the rapid quench flow instrument. Turn on the instrument, and after the main menu appears, type seven on the instrument keyboard to move the syringe driver to the home position.Open the buffer syringe position to load. Fill syringe C with quench solution, then, using a 10 milliliter syringe, fill the instrument buffer reservoirs A and B with buffer. Remove air bubbles by pushing plungers in and out.Change the knob positions to fire. Type two to adjust the position of the syringe driver bar. Press the adjust down button to lower the syringe until the buffer comes out the exit loop.Type escape to return to the main menu. To wash the tubing, attach the exit line to a vacuum. Change the sample knobs to flush.Set the knobs to the horizontal position to close the buffer syringe. Turn on the vacuum, dip the two flush loops in water for 10 seconds, and then dip in methanol for 10 seconds. Dry the loops by sucking air for about 15 seconds, or until dry.To load samples, change the sample knob position to load. Place solution A into a one milliliter syringe, plug the syringe into one of the sample load ports, in the same way, place solution B in the opposite sample load port. Begin the collection of time points by typing one on the main menu for a quench flow run.Enter the reaction time and press enter. The reaction loop number to use will be displayed. Switch to the required reaction loop.After washing the tube as demonstrated earlier, turn the sample knobs to load. Load the reaction mixes in the sample loops until just outside the central mixing compartment. Turn all the knobs to fire, and confirm that all knobs are in the correct position.Place a 1.5 milliliter tube onto the end of the exit loop. Press go to run the reaction. After repeating the tubing wash procedure, reload buffer syringes A and B until they hit the buffer syringe driver.Repeat the steps for running the reaction for each desired time point, with one exception, do not load solution B when quenching the zero time point control. When the sample collection is finished, enter escape to return to the main menu. Press seven to return the buffer syringe driver to its home position.Start the instrument clean up by removing the reaction mixture syringes, turn the sample loading knob to the load position. Under vacuum, wash each sample loop with 10 milliliters each of sodium hydroxide, phosphoric acid, and methanol. Press down on all three syringe plungers to retrieve the reaction and quench buffers.Wash the syringes with five milliliters of water at least twice. Fill the syringes again with five milliliters of water, turn the knobs to the fire position, turn on the vacuum and bring down the driver via the keyboard command to remove the water. Wash the tubing as demonstrated earlier.Lastly, turn off the rapid quench flow instrument. The samples are subsequently analyzed as described in the text protocol. These results were obtained by manually sampling a primer synthesis reaction catalyzed by GP four under multiple turnover conditions.The labeled precursor CTP shows the highest mobility and migrates towards the bottom of the gel, followed by slower migrating species, corresponding to the dye and tetraribonucleotides synthesized by GP four. Depending on the length of incubation and template sequence context, additional labeled products corresponding to higher oligomers can be observed. When a rapid quench instrument is used to examine the reaction catalyzed by GP four in timescales in the range of seconds down to a few ms, it is evident that primer accumulation is not linear, but displays a biphasic profile indicated by the solid line.This suggests that product formation is rapid, and release is rate-limiting in the steady state. The dotted line represents a hypothetical progress curve that would result if primer formation by GP four did not proceed with a pre-steady state burst. A single-turnover primer synthesis experiment provides information about the formation and decay of intermediate species during the conversion of substrate to product.A time course of tetramer formation is shown by the gel image. The graph shows intermediate formation versus time. Once mastered, this technique can be done in 90 minutes, if it's performed properly.Don't forget that working with radioactive reagents can be extremely hazardous and precautions such as proper shielding should always be taken while performing this procedure.

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7.2K Görüntüleme.  Harvard Medical School. The overall goal of this procedure is to examine the kinetics of primer and lagging strand DNA synthesis. This method can help answer key questions in the DNA replication field, such as how primers form by DNA primase, are transferred to, and utilized by, DNA polymerase. The main advantage of this technique is that it allows the identification of key steps during Okazaki fragment synthesis.Since radioactive materials are used in this experiment, all institutional regulations regarding ...